Although it is well-established that many malignancies can be transplanted, there is little evidence to demonstrate that a pre-malignant disease, such as myelodysplastic syndrome (MDS), can be transplanted and subsequently undergo malignant transformation in vivo. We used NUP98-HOXD13 (NHD13) mouse bone marrow to demonstrate that MDS could be transferred by a long-term repopulating cell. Recipients of the NHD13 MDS bone marrow displayed all of the features typical of MDS, including peripheral blood cytopenias, dysplasia, and transformation to AML. Ineffective hematopoiesis was evident in competitive repopulation assays. When equivalent numbers of WT and NHD13 cells were transplanted, equivalent numbers of cells were detected in the bone marrow, but the proportion of NHD13 cells in the peripheral blood was markedly reduced. However, despite this ineffective hematopoiesis, the NHD13 cells inexorably overgrew the WT cells. Even when transplanted with a tenfold excess of WT cells, the NHD13 cells gradually out-competed WT cells. Limiting dilution experiments demonstrated that the frequency of the cell which could transmit myelodysplastic syndrome was 1/6,000-1/16,000 and that myelodysplastic syndrome was also transferable to secondary recipients as a pre-malignant condition. Transformation to acute myeloid leukemia (AML) in primary transplant recipients was generally delayed (46-49 weeks post transplant);however, six of ten secondary transplant recipients developed AML. Since we identified the myelodysplastic syndrome-initiating cell (M-IC) in the linneg BM population, further defined the M-IC cell in terms of cell surface markers. In normal hematopoiesis, the long term hematopoietic repopulating cell is contained within the LinnegSca1+Kit+ (LSK) population. Surprisingly, the LSK and LinnegSca1-Kit+ populations are both decreased in the NHD13 mice compared to WT controls. Therefore, we tested three subpopulations LinnegSca-Kit+, LinnegSca-Kit- and LinnegSca+Kit+ for their ability to transfer myelodysplastic syndrome into lethally irradiated WT recipients. Our experiments demonstrated that the M-IC resides in the LinnegSca+Kit+ long term repopulating compartment. One reason for the difficulty in developing effective treatments for myelodysplastic syndrome is that there are no myelodysplastic syndrome cell lines which can be used to model or study the disease;cell lines that are described as myelodysplastic syndrome cell lines are really AML cell lines which have evolved from patients with myelodysplastic syndrome. Although numerous investigators have attempted to develop xenograft models for myelodysplastic syndrome, these attempts have met with little success. Given that the NHD13 mice develop a highly penetrant myelodysplastic syndrome that closely resembles the human disease, we have begun studies to determine if these mice are a useful pre-clinical model for myelodysplastic syndrome. Our initial studies have used the DNA-methyltransferase inhibitor 5-azacytidine. Our pilot study included 3 groups of mice [NHD13 mice injected with 5-azacytidine (n=6), NHD13 mice injected with saline (n=4), and WT mice injected with 5-azacytidine (n=4)]. After 16 weeks of therapy, the results appeared promising, as the treated NHD13 mice showed a significant increase in hemoglobin compared to the saline treated NHD13 mice (2.21 +/- 1.47 g/dL vs 0.13 +/- 0.66 g/dL, p=0.02). Unfortunately, three of the NHD13 treated mice were found dead in the following two months, and we were not able to determine the cause of death for these mice. The three remaining mice had gradual decreases in hemoglobin over the next 12 weeks, and were close to baseline hemoglobin levels. Nonetheless the observation that these mice had stable hemoglobin levels after 28 weeks was encouraging. To determine whether we were achieving levels of 5-azacytidine adequate to cause cytosine demethylation, we examined global and gene-specific methylation status, in collaboration with Dr. J.P. Issa (M.D. Anderson); these experiments demonstrated hypermethylation in the NHD13 mice compared to controls, and partial reversal of the hypermethylation with 5-azacytidine treatment. Because the experiments discussed above used transgenic mice, effective treatment with 5-azacytidine could not replace the MDS bone marrow with completely normal (ie, wildtype or WT) bone marrow, since all of the bone marrow was transgenic. Therefore, in order to distinguish improvement in peripheral blood cytopenia due to differentiation of the MDS clone from elimination of the MDS clone, we have begun repeating the 5-azacytidine treatment on chimeric mice, that have both WT and NHD13 bone marrow. If the results are promising, we will test the effect of different 5-azacytidine dosing schedules. Despite the recent FDA approval of three drugs for MDS, the only curative option for patients with MDS is allogeneic hematopoietic stem cell transplantation (HSCT). Allogeneic HSCT has two essential components, high-dose cytotoxic chemo-radiotherapy, and an immune-mediated graft versus host (GVH), or graft versus tumor (GVT) effect . However, the relative contributions of high dose cytotoxic therapy and GVT are not well established in MDS. We propose to use transplantation of the NHD13 mice as a means to investigate this question. The goals of our initial experiments are two-fold. First, we plan to determine if NHD13 mice with MDS can enter a long term remission through the use of lethal irradiation followed by transplantation with WT syngeneic hematopoietic stem cells (HSC). If mice enter a long term remission through lethal irradiation and HSC transfer, we will conclude that long term disease remission in this model can be achieved via high dose cytotoxic therapy alone, and that the contribution of GVT is not required. Our second goal is to identify a radiation dose that is effective at inducing a brief or partial remission, but ineffective at inducing a long term remission. This dose schedule will then be used to investigate a role for GVT effect in the treatment of MDS. Remission will be defined as normal peripheral blood counts, and no evidence of host-derived hematopoiesis, for 26-52 weeks post BMT. The radiation dose schedule effective at inducing a brief or partial remission will be employed for mismatched HSC transplants that will allow for a GVT effect. C3H.SW mice are identical to C57Bl6 mice at the major histocompatibility loci, but have numerous mismatches at minor histocompatibility loci108. For this reason, transplantation of C3H.SW donor cells into C57Bl6 host mice has been used to study GVH and GVT108. We will transplant T-cell depleted or whole C3H.SW BMNC into irradiated NHD13 C57Bl6 mice. Mice will be evaluated at timed intervals as described above for engraftment and peripheral blood hematopoiesis. In addition, mice will be weighed weekly as a means of assessing GVH. Mice that develop clinical signs consistent with severe GVH (weight loss, ruffled fur, lethargy), without signs of leukemia or MDS will be euthanized and evaluated for histologic signs of GVH. Our collaborator, Dr. Terry Fry, has experience in generating GVH using the above model, and has agreed to assist us in these studies. In addition to the experiments outlined above, we have transferred NHD13 mice to colleagues at several academic institutions, and are in the process of licensing NHD13 mice in order for them to be sent to three separate biotech companies for pre-clinical studies. These colleagues have plans to treat NHD [summary truncated at 7800 characters]